P015 A study to demonstrate heteroresistance and tolerance to azoles in Candida tropicalis

POSTER SESSION 1, SEPTEMBER 21, 2022, 12:30 PM - 1:30 PM: OBJECTIVE: Candida tropicalis rarely exhibits a trailing growth phenomenon in in vitro antifungal susceptibility testing (AFST) of azoles. This phenomenon is considered as ‘sensitive’ as per current CLSI guidelines. This phenomenon has been linked to treatment failure and such isolates could either be heteroresistant (HR) or tolerant, which is known with bacteria. HR subtype (<1%) of an isogenic susceptible cell population grows at drug concentrations at least 8 times higher than the minimum inhibitory concentration (MIC). Tolerant cells (5%-90% of the population) endure antimicrobial treatment several times above MIC. Characterizing these isolates could help relate their clinical susceptibility profiles to the treatment outcome. The aim of the study was to differentiate heteroresistance and tolerance from true azole resistance in C. tropicalis clinical isolates. METHODS: A total of 247 clinical isolates of C. tropicalis collected over a period of 7 months (September 2021 through March 2022) from PGIMER, Chandigarh were included in the study. Identification was done by MALDI-TOF MS and antifungal susceptibility testing to fluconazole was performed by CLSI broth microdilution method (M27-A3). Isolates exhibiting trailing growth phenomena were considered for further characterization. Cells from the trailing growth of each isolate were sub-cultured onto yeast extract peptone dextrose (YPD) agar to observe for phenotypic variations with and without fluconazole. The area under the population analysis (PAP) curve (AUC) was performed to determine the degree of heteroresistance. Yeast cells within the range of 102-105 cells/ml were spot inoculated in six replicates each onto a gradient of fluconazole (0.125-256 μg/ml) in YPD agar and incubated at 35°C for 5 days to determine the viable colony forming units (CFU). RESULTS: Out of the 225 fluconazole susceptible C. tropicalis isolates, 10 (4.4%) were found to exhibit a trailing growth phenomenon. A dose-response relationship showed a multimodal population distribution in these isolates with varying degrees of heteroresistance as demonstrated by area under curve that ranged from 171-1331. The heterogeneous subpopulations sampled from the growth at the highest fluconazole concentration (64 μg/ml) exhibited a similar MIC (± one 2-fold difference) as that of the parent isolate when tested individually. The heterogeneity range that determines a fold difference breakpoint of the isolates varied from 16 to >256. CONCLUSION: An isogenic population of cells under the effect of fluconazole could give rise to phenotypically different subpopulations. With repeated exposure to the azole drugs, these seemingly susceptible isolates can emerge as fully resistant population. Clinically, the implications include relapse, treatment failure, and persistent chronic infection, owing to which this phenomenon needs attention. Current CLSI guidelines do not provide any criteria to separately classify these isolates from the susceptible and resistant varieties. Hence a definitive cut-off is warranted to identify the HR and tolerant subtypes. The AUC-PAP method could be refined further for discriminating heteroresistance from true resistance.